All-trans- and 9-cis-retinoic acid are active retinoids for regulating expression of retinoid responsive genes, serving as ligands for two classes of ligand-dependent transcription factors, the retinoic acid receptors and retinoid X receptors. Little is known, however, regarding 9-cis-retinoic acid formation. We have obtained a 1.4-kilobase cDNA clone from a normalized human breast tissue library, which when expressed in CHO cells encodes a protein that avidly catalyzes oxidation of 9-cis-retinol to 9-cis-retinaldehyde. This protein also catalyzes oxidation of 13-cis-retinol at a rate approximately 10% of that of the 9-cis isomer but does not catalyze all-trans-retinol oxidation. NAD+ was the preferred electron acceptor for oxidation of 9-cis-retinol, although NADP+ supported low rates of 9-cis-retinol oxidation. The rate of 9-cis-retinol oxidation was optimal at pHs between 7.5 and 8. Sequence analysis indicates that the cDNA encodes a protein of 319 amino acids that resembles members of the short chain alcohol dehydrogenase protein family. mRNA for the protein is most abundant in human mammary tissue followed by kidney and testis, with lower levels of expression in liver, adrenals, lung, pancreas, and skeletal muscle. We propose that this cDNA encodes a previously unknown stereospecific enzyme, 9-cis-retinol dehydrogenase, which probably plays a role in 9-cis-retinoic acid formation.
Retinoids (vitamin A and its analogs) are essential dietary substances that are needed by mammals for reproduction, normal embryogenesis, growth, vision, and maintaining normal cellular differentiation and the integrity of the immune system (1–5). Within cells, retinoids regulate gene transcription acting through ligand-dependent transcription factors, the retinoic acid receptors (RARs)1, and the retinoid X receptors (RXRs) (6,7). All-trans-retinoic acid binds only to RARs with high affinity, whereas its 9-cis isomer binds with high affinity to both RARs and RXRs. The actions of all-trans- and 9-cis-retinoic acid in regulating cellular responses are distinct and not interchangeable.
In contrast to the great explosion of information regarding the actions of retinoid receptors in regulating gene transcription, information regrading how the abundant precursor retinol is physiologically activated to form the ligands needed to activate retinoid receptors is only slowly emerging (see Refs. 8 and 9 for recent reviews). It is clear that the pathway for conversion of retinol to retinoic acid involves first the oxidation of retinol to retinaldehyde and then the oxidation of retinaldehyde to retinoic acid. Numerous enzymes that are able to catalyze either retinol or retinaldehyde oxidation have been identified, purified, and/or characterized (8–10). These enzymes are members of four distinct families: the alcohol dehydrogenases, the short chain alcohol dehydrogenases, the aldehyde dehydrogenases, and cytochrome P-450s (8–10). At present, the most attention has focused on enzymes responsible for the oxidation of all-trans-retinol to all-trans-retinaldehyde (11–15). Several recent reports have indicated that both alcohol dehydrogenases and short chain alcohol dehydrogenases may be responsible for catalyzing all-trans-retinol oxidation (11–15), but the exact in vivo roles of each of these dehydrogenases in all-trans-retinoic acid formation remains controversial (8).
9-cis-Retinoic acid has been reported to be present in mammalian tissues and cells (16–18), but it has not been convincingly established how 9-cis-retinoic acid is formed within tissues and cells. Urbach and Rando have reported that liver microsomes can nonenzymatically catalyze the isomerization of all-trans-retinoic acid to the 9-cis isomer (19). Others have demonstrated that 9-cis-5-carotene can be converted to 9-cis-retinoic acid within rat tissues (20). However, this latter pathway cannot be an essential one for 9-cis-retinoic acid formation because rats maintained on a β-carotene-free purified diet containing only retinol as a precursor for retinoic acid formation are normal. In this communication, we report the characterization of a cDNA clone for a novel human enzyme that we have designated 9-cis-retinol dehydrogenase (9cRDH) and that catalyzes in a stereospecific manner the oxidation of 9-cis-retinol to 9-cis-retinaldehyde, a first enzymatic step needed for 9-cis-retinoic acid formation. Because it has been established that9-cis-retinaldehyde can be further oxidized to 9-cis-retinoic acid by abundant tissue retinaldehyde dehydrogenases (21–23), it is possible that 9cRDH catalyzes a key oxidation step in the formation of 9-cis-retinoic acid.